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1.
Postepy Biochem ; 65(3): 173-182, 2019 10 01.
Artigo em Polonês | MEDLINE | ID: mdl-31643164

RESUMO

Endoribonuclease III Dicer plays a crucial role in the biogenesis of small regulatory RNAs, such as microRNAs (miRNAs) and small inter­fering RNAs (siRNAs). However, this is not the only role that Dicer plays in cells. For example, it has been shown that Dicer is involved in processing of diverse classes of RNA, including tRNA and snoRNA, cleavage of repeat-element-derived RNAs, and maintenance of genome integrity. Dicer has also been found to participate in the chromosome fragmentation during apoptosis or in the inflammatory processes. More­over, a recent discovery of Dicer-binding passive sites in mRNAs and long non-coding RNAs, and its putative nucleic acid chaperone activity, has pointed out a novel regulatory role of the enzyme. Here we focus on human Dicer and review its structure and function including recent findings on miRNA-independent roles and their impact on cell biology.


Assuntos
Ribonuclease III/química , Ribonuclease III/metabolismo , Fragmentação do DNA , Humanos , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Processamento Pós-Transcricional do RNA , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/biossíntese , Pequeno RNA não Traduzido/metabolismo
2.
Microbiol Res ; 229: 126319, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479952

RESUMO

Methionine is critical for variety of metabolic processes in biological organisms, acting as a precursor or intermediate for many final products. The last step for the synthesis of methionine is the methylation of homocysteine, which is catalyzed by MetE. Here, we use Salmonella enterica serovar Typhimurium LT2 to study the regulation of the metE+ gene by an anaerobically induced small non-coding RNA-FnrS, the expression of which is strictly dependent on the anaerobic regulator-FNR. The MetE-HA protein was expressed at an increased level in the fnrS- and hfq- deficient strains under anaerobic conditions. The Hfq protein is predicted to stabilize the binding between small RNA(s) and their target mRNA(s). A transcriptional (op) and translational (pr) metE::lacZ fusion gene were separately constructed, with the metE+-promoter fused to a lacZ reporter gene. In an anaerobic environment, the metE::lacZ (pr) fusion gene and reverse transcription-PCR identified that FnrS and/or FNR negatively regulate metE+ mRNA levels in the rich media. Analysis of FnrS revealed a sequence complementary to the 5' mRNA translational initiation region (TIR) of the metE+ gene. Mutation(s) predicted to disrupt base pairing between FnrS and metE+ TIR were constructed in fnrS, and most of those resulted in the loss of repressive activity. When compensatory mutation(s) were made in metE+ 5' TIR to restore base pairing with FnrS, the repressive regulation was completely restored. Therefore, in this study, we identified that in anaerobic phase, there is a repression of metE+ gene expression by FnrS and that base-paring, between both expressive transcripts, plays an important role for this negative regulation.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Metiltransferases/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Pequeno RNA não Traduzido/genética , Salmonella typhimurium/enzimologia , Proteínas de Bactérias/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação Enzimológica da Expressão Gênica , Metiltransferases/química , Metiltransferases/metabolismo , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo
3.
Chem Pharm Bull (Tokyo) ; 67(8): 877-883, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31366836

RESUMO

The 4-vinylpyrimidin-2-one nucleoside (T-vinyl) forms a cross-link with the RNA containing uracil at the complementary site at a high reaction rate. To obtain the stable T-vinyl derivative so that its reactivity is protected until it access to the target site, several derivatives were investigated, and the 2-thiopyridinyl- and 2-thiopyrimidinyl T-vinyl derivatives were determined to be good candidates. The 2-thiopyrimidinyl T-vinyl derivative was found to more efficiently cross-link with mRNA albeit having a better stability than the 2-thiopyridinyl T-vinyl derivative. The investigation using the luciferase (Luc) mRNA, the synthetic mRNA and non-cellular translation system revealed that the translation is terminated at the end of the cross-linked duplex between the mRNA and the oligoribonucleotide (ORN). Thus, the 2-thiopyrimidinyl T-vinyl derivative has successfully demonstrated both a good stability and high efficiency for the cross-linking reaction, and expanded its applicability in biological applications.


Assuntos
Reagentes para Ligações Cruzadas/química , Nucleosídeos/química , Oligorribonucleotídeos/química , RNA Mensageiro/química , Compostos de Vinila/química , Reagentes para Ligações Cruzadas/síntese química , Estrutura Molecular , Nucleosídeos/síntese química , Compostos de Vinila/síntese química
4.
Genome Biol ; 20(1): 156, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387610

RESUMO

BACKGROUND: Methylation of nucleotides, notably in the forms of 5-methylcytosine (5mC) in DNA and N6-methyladenosine (m6A) in mRNA, carries important information for gene regulation. 5mC has been elucidated to participate in the regulation of fruit ripening, whereas the function of m6A in this process and the interplay between 5mC and m6A remain uncharacterized. RESULTS: Here, we show that mRNA m6A methylation exhibits dynamic changes similar to DNA methylation during tomato fruit ripening. RNA methylome analysis reveals that m6A methylation is a prevalent modification in the mRNA of tomato fruit, and the m6A sites are enriched around the stop codons and within the 3' untranslated regions. In the fruit of the ripening-deficient epimutant Colorless non-ripening (Cnr) which harbors DNA hypermethylation, over 1100 transcripts display increased m6A levels, while only 134 transcripts show decreased m6A enrichment, suggesting a global increase in m6A. The m6A deposition is generally negatively correlated with transcript abundance. Further analysis demonstrates that the overall increase in m6A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene SlALKBH2, which is regulated by DNA methylation. Interestingly, SlALKBH2 has the ability to bind the transcript of SlDML2, a DNA demethylase gene required for tomato fruit ripening, and modulates its stability via m6A demethylation. Mutation of SlALKBH2 decreases the abundance of SlDML2 mRNA and delays fruit ripening. CONCLUSIONS: Our study identifies a novel layer of gene regulation for key ripening genes and establishes an essential molecular link between DNA methylation and mRNA m6A methylation during fruit ripening.


Assuntos
Adenosina/análogos & derivados , Regulação da Expressão Gênica de Plantas , Lycopersicon esculentum/genética , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Metilação de DNA , Retículo Endoplasmático/enzimologia , Frutas/genética , Frutas/metabolismo , Regulação Enzimológica da Expressão Gênica , Lycopersicon esculentum/enzimologia , Lycopersicon esculentum/metabolismo , Metilação , Mutação , Motivos de Nucleotídeos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
5.
Genome Biol ; 20(1): 162, 2019 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399036

RESUMO

BACKGROUND: General translational cis-elements are present in the mRNAs of all genes and affect the recruitment, assembly, and progress of preinitiation complexes and the ribosome under many physiological states. These elements include mRNA folding, upstream open reading frames, specific nucleotides flanking the initiating AUG codon, protein coding sequence length, and codon usage. The quantitative contributions of these sequence features and how and why they coordinate to control translation rates are not well understood. RESULTS: Here, we show that these sequence features specify 42-81% of the variance in translation rates in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Mus musculus, and Homo sapiens. We establish that control by RNA secondary structure is chiefly mediated by highly folded 25-60 nucleotide segments within mRNA 5' regions, that changes in tri-nucleotide frequencies between highly and poorly translated 5' regions are correlated between all species, and that control by distinct biochemical processes is extensively correlated as is regulation by a single process acting in different parts of the same mRNA. CONCLUSIONS: Our work shows that general features control a much larger fraction of the variance in translation rates than previously realized. We provide a more detailed and accurate understanding of the aspects of RNA structure that directs translation in diverse eukaryotes. In addition, we note that the strongly correlated regulation between and within cis-control features will cause more even densities of translational complexes along each mRNA and therefore more efficient use of the translation machinery by the cell.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas , RNA Mensageiro/química , Animais , Arabidopsis/genética , Humanos , Camundongos , Modelos Genéticos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
7.
Nat Chem Biol ; 15(9): 865-871, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31383972

RESUMO

RNA modification in the form of N6-methyladenosine (m6A) regulates nearly all the post-transcriptional processes. The asymmetric m6A deposition suggests that regional methylation may have distinct functional consequences. However, current RNA biology tools do not distinguish the contribution of individual m6A modifications. Here we report the development of 'm6A editing', a powerful approach that enables m6A installation and erasure from cellular RNAs without changing the primary sequence. We engineered fusions of CRISPR-Cas9 and a single-chain m6A methyltransferase that can be programmed with a guide RNA. The resultant m6A 'writers' allow functional comparison of single site methylation in different messenger RNA regions. We further engineered m6A 'erasers' by fusing CRISPR-Cas9 with ALKBH5 or FTO to achieve site-specific demethylation of RNAs. The development of programmable m6A editing not only expands the scope of RNA engineering, but also facilitates mechanistic understanding of epitranscriptome.


Assuntos
Adenosina/análogos & derivados , Sistemas CRISPR-Cas , Edição de Genes/métodos , Metiltransferases/metabolismo , RNA Mensageiro/metabolismo , Adenosina/química , Adenosina/metabolismo , Sequência de Bases , Linhagem Celular , Humanos , Metiltransferases/classificação , RNA Mensageiro/química , RNA Mensageiro/genética
8.
BMC Bioinformatics ; 20(1): 379, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286861

RESUMO

BACKGROUND: Unsupervised machine learning methods (deep learning) have shown their usefulness with noisy single cell mRNA-sequencing data (scRNA-seq), where the models generalize well, despite the zero-inflation of the data. A class of neural networks, namely autoencoders, has been useful for denoising of single cell data, imputation of missing values and dimensionality reduction. RESULTS: Here, we present a striking feature with the potential to greatly increase the usability of autoencoders: With specialized training, the autoencoder is not only able to generalize over the data, but also to tease apart biologically meaningful modules, which we found encoded in the representation layer of the network. Our model can, from scRNA-seq data, delineate biological meaningful modules that govern a dataset, as well as give information as to which modules are active in each single cell. Importantly, most of these modules can be explained by known biological functions, as provided by the Hallmark gene sets. CONCLUSIONS: We discover that tailored training of an autoencoder makes it possible to deconvolute biological modules inherent in the data, without any assumptions. By comparisons with gene signatures of canonical pathways we see that the modules are directly interpretable. The scope of this discovery has important implications, as it makes it possible to outline the drivers behind a given effect of a cell. In comparison with other dimensionality reduction methods, or supervised models for classification, our approach has the benefit of both handling well the zero-inflated nature of scRNA-seq, and validating that the model captures relevant information, by establishing a link between input and decoded data. In perspective, our model in combination with clustering methods is able to provide information about which subtype a given single cell belongs to, as well as which biological functions determine that membership.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Neurais (Computação) , RNA Mensageiro/química , Análise de Sequência de RNA/métodos , Aprendizado de Máquina não Supervisionado , Análise por Conglomerados , RNA Mensageiro/metabolismo , Análise de Célula Única
9.
Chem Commun (Camb) ; 55(61): 8959-8962, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290487

RESUMO

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Evolução Molecular Direcionada/métodos , Proteínas Nucleares/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Peptídeos/metabolismo , Engenharia de Proteínas/métodos , Fatores de Transcrição/metabolismo , Alquilação , Sequência de Aminoácidos , Ciclização , Cisteína/química , Hidrocarbonetos Bromados/química , Biblioteca de Peptídeos , Peptídeos/química , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/química
10.
Nature ; 571(7765): 424-428, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292544

RESUMO

N6-methyladenosine (m6A) is the most prevalent modified nucleotide in mRNA1,2, with around 25% of mRNAs containing at least one m6A. Methylation of mRNA to form m6A is required for diverse cellular and physiological processes3. Although the presence of m6A in an mRNA can affect its fate in different ways, it is unclear how m6A directs this process and why the effects of m6A can vary in different cellular contexts. Here we show that the cytosolic m6A-binding proteins-YTHDF1, YTHDF2 and YTHDF3-undergo liquid-liquid phase separation in vitro and in cells. This phase separation is markedly enhanced by mRNAs that contain multiple, but not single, m6A residues. Polymethylated mRNAs act as a multivalent scaffold for the binding of YTHDF proteins, juxtaposing their low-complexity domains and thereby leading to phase separation. The resulting mRNA-YTHDF complexes then partition into different endogenous phase-separated compartments, such as P-bodies, stress granules or neuronal RNA granules. m6A-mRNA is subject to compartment-specific regulation, including a reduction in the stability and translation of mRNA. These studies reveal that the number and distribution of m6A sites in cellular mRNAs can regulate and influence the composition of the phase-separated transcriptome, and suggest that the cellular properties of m6A-modified mRNAs are governed by liquid-liquid phase separation principles.


Assuntos
Adenosina/análogos & derivados , Compartimento Celular , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Adenosina/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Humanos , Metilação , Metiltransferases/deficiência , Camundongos , Transição de Fase , RNA Mensageiro/análise , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Estresse Fisiológico
11.
Nat Biotechnol ; 37(7): 803-809, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31267113

RESUMO

The ability to predict the impact of cis-regulatory sequences on gene expression would facilitate discovery in fundamental and applied biology. Here we combine polysome profiling of a library of 280,000 randomized 5' untranslated regions (UTRs) with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5' UTRs that accurately direct specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,212 truncated human 5' UTRs and 3,577 naturally occurring variants and show that the model predicts ribosome loading of these sequences. Finally, we provide evidence of 45 single-nucleotide variants (SNVs) associated with human diseases that substantially change ribosome loading and thus may represent a molecular basis for disease.


Assuntos
Regiões 5' não Traduzidas , Biossíntese de Proteínas , RNA Mensageiro/genética , Sequência de Bases , Regulação da Expressão Gênica , Humanos , Modelos Genéticos , Pseudouridina/análogos & derivados , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Ribossomos
12.
Adv Mater ; 31(33): e1902575, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31215123

RESUMO

A main challenge to broaden the biomedical application of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat (CRISPR) associated protein 9) genome editing technique is the delivery of Cas9 nuclease and single-guide RNA (sgRNA) into the specific cell and organ. An effective and very fast CRISPR/Cas9 genome editing in vitro and in vivo enabled by bioreducible lipid/Cas9 messenger RNA (mRNA) nanoparticle is reported. BAMEA-O16B, a lipid nanoparticle integrated with disulfide bonds, can efficiently deliver Cas9 mRNA and sgRNA into cells while releasing RNA in response to the reductive intracellular environment for genome editing as fast as 24 h post mRNA delivery. It is demonstrated that the simultaneous delivery of Cas9 mRNA and sgRNA using BAMEA-O16B knocks out green fluorescent protein (GFP) expression of human embryonic kidney cells with efficiency up to 90%. Moreover, the intravenous injection of BAMEA-O16B/Cas9 mRNA/sgRNA nanoparticle effectively accumulates in hepatocytes, and knocks down proprotein convertase subtilisin/kexin type 9 level in mouse serum down to 20% of nontreatment. The leading lipid nanoparticle, BAMEA-O16B, represents one of the most efficient CRISPR/Cas9 delivery nanocarriers reported so far, and it can broaden the therapeutic promise of mRNA and CRISPR/Cas9 technique further.


Assuntos
Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Lipídeos/química , Nanopartículas/química , RNA Guia/química , RNA Mensageiro/química , Animais , Transporte Biológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes/métodos , Técnicas de Transferência de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Oxirredução , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , RNA Guia/administração & dosagem , RNA Mensageiro/administração & dosagem
13.
Nat Commun ; 10(1): 2569, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189880

RESUMO

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb ( http://SynMICdb.dkfz.de ), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure.


Assuntos
Bases de Dados de Ácidos Nucleicos , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Mutação Silenciosa/genética , Conjuntos de Dados como Assunto , Humanos , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Dobramento de RNA/genética , Processamento de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética
14.
Microb Pathog ; 132: 275-281, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078709

RESUMO

Toxoplasma gondii is an obligate intracellular parasite that causes one of the most common parasitic infections in humans and other warm-blooded animals. Currently, there are no effective treatments for inhibiting the formation of chronic tissue cysts in infected hosts. Thus, the development of a vaccine to protect against toxoplasmosis is an attractive option for avoiding infection. The aim of this study was to design an epitope-based vaccine for T. gondii. In the present study, an in silico approach was used to predict and analyze B-cell and T-cell epitopes and the transmembrane domain of proteins SAG1, MIC3, and ROP8. We also predicted the antigenicity, allergenicity, secondary and tertiary structures, and physicochemical characteristics of a chimeric protein. Next, codon optimization and mRNA structure prediction were conducted using bioinformatics tools, and the designed construct was chemically synthesized and cloned into the pET28a vector. SAG1 (amino acid positions 85-235), MIC3 (30-180), and ROP8 (85-185) were found to have several strong immunodominant epitopes that were joined with a rigid linker A(EAAAK)2A. Although the resultant protein called MRS (MIC3, ROP8, and SAG1) did not turn out to be an allergen, its antigenicity was estimated to be 0.7983. Additionally, MRS was selected as the best vaccine candidate on the basis of its secondary and tertiary structures. The number of amino acids, molecular weight, and numbers of negatively and positively charged residues of MRS were 427 and 45,661.31 Da, 45, and 50, respectively. ΔG of the best-predicted structure was -413.0 kcal/mol, and the first nucleotides at the 5' end did not form a stable hairpin or pseudoknot. Finally, successful expression and verification of the expressed MRS protein showed that in silico analysis was almost accurate. This vaccine candidate selected by in silico tools should be validated in experimental studies.


Assuntos
Antígenos de Protozoários/imunologia , Imunogenicidade da Vacina/imunologia , Vacinas Protozoárias/imunologia , Proteínas Recombinantes de Fusão/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/genética , Biologia Computacional , Simulação por Computador , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Expressão Gênica , Humanos , Modelos Moleculares , Conformação Proteica , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA Mensageiro/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Toxoplasmose/imunologia , Toxoplasmose/prevenção & controle
15.
Yi Chuan ; 41(5): 404-412, 2019 May 20.
Artigo em Chinês | MEDLINE | ID: mdl-31106776

RESUMO

N 6-methyladenosine (m 6A) is a prevalent modification of RNA in eukaryotes and plays an important role in the process of mRNA translocation, stabilization and translation. m 6A exerts different influences on the viral replication cycle, and both viral replication and host immune response to the virus are affected by m 6A. In this review, we summarize recent studies on the mechanism of m 6A modification and its effects on viral replication and host immune response, in order to provide a reference for epigenetic regulation in the viral life cycle.


Assuntos
Adenosina/análogos & derivados , Epigênese Genética , RNA Mensageiro/química , Replicação Viral , Adenosina/química , RNA Mensageiro/genética , Vírus
16.
RNA ; 25(8): 1038-1046, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31064786

RESUMO

Visualization of gene expression at single RNA molecular level represents a great challenge to both imaging technologies and molecular engineering. Here we show a single molecule chromogenic in situ hybridization (smCISH) assay that enables counting and localizing individual RNA molecules in fixed cells and tissue under bright-field microscopy. Our method is based on in situ padlock probe assays directly using RNA as a ligation template and rolling circle amplification combined with enzyme catalyzed chromogenic reaction for amplification product visualization. We show potential applications of our method by detecting gene expression variations in single cells, subcellular localization information of expressed genes, and gene expression heterogeneity in formalin-fixed, paraffin-embedded tissue sections. This facile and straightforward method can in principle be applied to any type of RNA molecules in different samples.


Assuntos
Compostos Cromogênicos/química , RNA Mensageiro/análise , Imagem Individual de Molécula/métodos , Animais , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , RNA Mensageiro/química , Inclusão do Tecido , Fixação de Tecidos
17.
Gene ; 707: 231-238, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31063797

RESUMO

Recent developments in the field of the messenger RNA and its advantages versus DNA have led to a renewed interest in mRNA-based technologies. Despite its advantages, mRNA therapy has a number of drawbacks including low amount of mRNA production, short-term existence of mRNA and mRNA-mediated protein within the cell, severe mRNA cytotoxicity, and immune response activation following mRNA transfection. Here, we applied untranslated regions of human beta-globin to increase the stability and translation efficiency of a destabilized GFP mRNA. In order to suppress the innate immune response, which is the main barrier of mRNA therapy, we used the vaccinia virus derived capping enzyme and substituted standard nucleotides with modified nucleotides. At the end, the Kozak sequence of human beta-globin was replaced with the strongest sequence for the further improvement of mRNA translation. Overall, these modifications with native Kozak (K1) sequence of human beta-globin enhanced the stability of destabilized GFP mRNA up to 48 h and no increase in the level of interferon-α and -ß was found. The GFP expression of mRNA with modified Kozak (K2) sequence initiated earlier than mRNA and plasmid DNA with K1 sequence. In contrast to mRNA with K1 sequence, the cells containing mRNA with K2 sequence remained positive for GFP expression up to 72 h post-transfection. Interestingly, transfection efficiency and mean fluorescence intensity (MFI) of mRNA with K2 sequence were higher than mRNA and plasmid DNA with K1 sequence. Taken together, these results provide valuable information for the optimization of mRNA stability and translation. Therefore, the methods used in the current study can successfully be applied for reprogramming, gene editing, trans-differentiation, tumour therapy, and gene therapy.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Globinas beta/genética , Regiões 3' não Traduzidas , Animais , Regulação da Expressão Gênica , Células HEK293 , Humanos , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/uso terapêutico , Transfecção , Pesquisa Médica Translacional
18.
BMC Genomics ; 20(1): 393, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113365

RESUMO

BACKGROUND: The behavioural transition from nurses to foragers in honey bees is known to be affected by intrinsic and extrinsic factors, including colony demography, hormone levels, brain chemistry and structure, and gene expression in the brain. However, the molecular mechanism underlying this behavioural transition of honey bees is still obscure. RESULTS: Through RNA sequencing, we performed a comprehensive analysis of lncRNAs and mRNAs in honey bee nurses and foragers. Nurses and foragers from both typical colonies and single-cohort colonies were used to prepare six libraries to generate 49 to 100 million clear reads per sample. We obtained 6863 novel lncRNAs, 1480 differentially expressed lncRNAs between nurses and foragers, and 9308 mRNAs. Consistent with previous studies, lncRNAs showed features distinct from mRNAs, such as shorter lengths, lower exon numbers, and lower expression levels compared to mRNAs. Bioinformatic analysis showed that differentially expressed genes were mostly involved in the regulation of sensory-related events, such as olfactory receptor activity and odorant binding, and enriched Wnt and FoxO signaling pathways. Moreover, we found that lncRNAs TCONS_00356023, TCONS_00357367, TCONS_00159909 and mRNAs dop1, Kr-h1 and HR38 may play important roles in behavioural transition in honey bees. CONCLUSION: This study characterized the expression profile of lncRNAs in nurses and foragers and provided a framework for further study of the role of lncRNAs in honey bee behavioural transition.


Assuntos
Abelhas/genética , RNA Longo não Codificante/metabolismo , Animais , Abelhas/metabolismo , Abelhas/fisiologia , Comportamento Animal , Perfilação da Expressão Gênica , RNA Longo não Codificante/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
19.
Biochimie ; 163: 21-32, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31075282

RESUMO

Huntington's diseases (HD) is a very devastating disease caused by r(CAG) expansion in HTT gene, encoding the huntingtin protein. r(CAG) expansion causes disease via multiple pathways including, 1) loss of normal protein function like sequestration of RNA binding protein such as Muscleblind-like (MBNL) and nucleolin, 2) Gain of function for mutant proteins and 3) repeat-associated non-ATG (RAN) translation; in which expanded r(CAG) translates into toxic poly glu, poly ser, or poly ala without the use of any canonical start codon. Herein, we have rationally designed and synthesized a unique class of pyridocoumarin derivatives that target the r(CAG)exp involved in HD and spinocerebellar ataxia (SCA) pathogenesis. Notably, compounds 3 and 15 showed higher affinity (nanomolar Kd) and selectivity for diseased r(CAG)exp RNA compared to regular duplex AU-paired RNA. Interestingly, both scaffolds are cell permeable, exhibit low toxicity to healthy fibroblast cells and are also capable of reducing the level of poly Q aggregation in cellular models. Indeed, our current study offers promising facet for selectively targeting repeats containing RNAs that cause severe diseases like HD and SCAs.


Assuntos
Cumarínicos/química , Doença de Huntington/tratamento farmacológico , Proteínas Mutantes/genética , RNA Mensageiro/química , Ataxias Espinocerebelares/tratamento farmacológico , Células Cultivadas , Cumarínicos/farmacologia , Cumarínicos/uso terapêutico , Desenho de Drogas , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Cinética , Simulação de Acoplamento Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/efeitos dos fármacos , Ataxias Espinocerebelares/metabolismo , Expansão das Repetições de Trinucleotídeos
20.
Eur J Cell Biol ; 98(2-4): 94-102, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053368

RESUMO

Competing endogenous RNAs (ceRNAs) are RNAs that share common miRNA binding sites and compete with each other for the miRNA association at these sites. The observation of this phenomenon in the cells altered the view of the miRNA target RNAs from molecules that are passively controlled by miRNAs to molecules that also modulate the miRNAs activity. In this review, we build a general profile of ceRNAS characteristics in order to facilitate ceRNAs identification by researchers. The information summarized here contains an actualized list of previously reported ceRNAs and classes of RNAs that can participate in this type of interaction, the expression behavior and characteristics of ceRNAs and miRNAs in the context of competition, the influence of the shared MREs/miRNAs numbers and the miRNA binding strength on the competition, reports on competition between RNAs in different subcellular localizations and the concept that ceRNAs may form a huge regulatory network in the cell.


Assuntos
Redes Reguladoras de Genes , Inativação Gênica , MicroRNAs/genética , RNA Mensageiro/genética , Animais , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo
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